Hydrogen peroxide (H2O2) is one of the reactive oxygen species (ROS) and a secondary messenger that plays an important role in many signaling pathways in living organisms. ROS species generally further include for example singlet oxygen, superoxide radical, hydroxyl radical, and hypochlorite. These species are generated and localized in organelles such as mitochondria.
Whilst controlled generation of H2O2 is beneficial to cell fitness, mal-regulation on the H2O2 level is associated with the pathogenesis of various diseases. It is related to oxidative stress and causes oxidative damage to intracellular biomolecules. Especially, the H2O2 level in the mitochondria is of particular importance because abnormal production and accumulation of H2O2 at this organelle has been linked to serious diseases such as cancer, Alzheimer's, Parkinson's and Huntington's diseases.
Existing and commercially available imaging reagents for H2O2 are fluorescent organic dyes. Although they show strong fluorescence, their use is limited by several factors including the fact that they do not allow for a selective detection of H2O2 over other types of ROS species, i.e. they can only detect the presence of total ROS species.
Moreover, they do not allow for an organelle specific detection of H2O2, i.e. these imaging reagents do not target on specific organelles so that it is difficult to determine which part of the cell/organism has gained an increase in H2O2 level.
Still further, these imaging reagents have a low photo-stability which does usually not allow prolonged irradiation usually required for real-time monitoring of H2O2 levels in cells. Additionally, the emission of commercially used organic dyes is fluorescence in nature which is associated with a short lifetime, namely in the nanosecond timescale, which is, thus, not sufficient for time-resolved and time-gated detection and microscopy such as fluorescence-lifetime imaging microscopy (FLIM). Moreover, existing sensors for H2O2, namely fluorescent organic dyes, suffer from high photobleaching rates, substantial and undesired self-quenching, and high pH dependence.
Accordingly, there remains a strong need for biosensors with increased selectivity towards ROS-species especially increased selectivity towards H2O2, suitable photo-stability, and suitable emission behavior which, for example, allow for real-time monitoring and time-resolved and time-gated detection of specific ROS species in specific organelles.